Figure 3.

NP1 knockdown increases [Ca²⁺]_i and the amplitude of spontaneous intracellular Ca²⁺ oscillations. Cortical cultured neurons were transduced with shNP1 to knockdown NP1 expression, with pWPI-NP1 to overexpress NP1, or with shRdm as control. A–C, Intracellular Ca²⁺ concentration was measured in the soma of mature neurons with Fura-2. A, Representative images of Fura-2 fluorescence pseudo-colored according to intensity in cortical cell cultures after gain and loss of NP1 function. B, Spontaneous [Ca²⁺]_i oscillations. Values are mean ± SEM of 52 neurons for shRdm (dotted line), 52 neurons for shNP1 (solid line), and 36 neurons for pWPI-NP1 (dashed line) from at least n = 4 independent cultures. Mean [Ca²⁺]_i of shNP1 is significantly higher than pWPI-NP1 and shRdm (p < 0.001, independent t test). NP1 knockdown significantly increased the amplitude of [Ca²⁺]_i oscillations compared with control or NP1 overexpressing neurons (p < 0.001, independent t test). NP1 overexpression significantly reduced basal [Ca²⁺]_i. C, Spontaneous [Ca²⁺]_i oscillations induced by knockdown of NP1 are abolished after addition of TTX (5 μm; 23 neurons).